EMortal™ is a hydrolyzed pea protein, a peptone. Actually EMortal™ is a small peptide mixture extracted from Pea( Pisum sativum L.). The peptides are extracted through a process that uses no harmful chemicals. The activity of EMortal™ on the stem cells has been proven by scientific research and a clinical trial.

Since the peptides have no antigens, they are not recognized as foreign by the host body because of their size and there are no allergic reactions. The use of plant origin to stimulate the stem cells also eliminates ethical considerations, since it causes no harm to the animal or involvement with human tissues.

Cell culture technology is used in virtually all fields of biomedical research and testing. We have developed a stem cell culture model for finding new compounds to activate stem cell proliferation. The effects of TGF-β1- elevating agents on the activity of TGF-β1 promoter are studied using the luciferase(LUC)-reporter-gene system.

The cytokine TGF-β1 plays an important role in regulating the wound healing and tissue repair process; and aberration of its action has been implicated in several pathological processes including cancer, autoimmune disease and fibrosis. Specifically in the skin, TGF-β1 induces proliferation of dermal fibroblasts and generates increased levels of Hyaluronan(HA), matrix polysaccharide. In the skin, the extracellular matrix(ECM) is composed of HA and other sulfated GAGs, combined with collagen and elastin. Large amounts of water are held in the ECM. When elastin is not bathed in water, it becomes dry and brittle, thus the look of dry, brittle, wrinkled skin. HA also influence fibroblast proliferation and thereby affect wound healing outcomes.

In skin keratinocytes, TGF-β1 has been shown to inhibit growth and to rapidly reduce protooncogene, c-myc expression. TGF-β1 is a potent tumor suppressor.

Therefore TGF-β1 activators are very useful for skin care products and potentially become stem cell activators.

Using this culture model we have found a few TGF-β1 activators including EMortal™. Stimulation of the transfected stem cells with EMortal™ enhanced both TGF-β1 dependent transcription of luciferase gene and cell growth.

Since the early days of cell and tissue culture, animal serum has been added to the culture media as a source of nutrients. Sometimes special experiments are used basal medium without serum to elucidate function of specific chemicals on cell physiology including cell proliferation.

We used a minimally supplemented, serum-free, stem cell culture system to confirm that EMortal™ has got activity on the stem cell proliferation. In this study, EMortal™ increased cell growth rate significantly by approximately 50% in the cell culture without serum in figure.

The effect was more confirmed by the results of the Click-iT TM EdU flow cytometry assay of the stem cells. As shown in Figure, the median value of the group treated with EMortal™ was shifted to the right when compared with the serum free group(7.23 versus 3.08). Taken together, these findings suggest that EMortal™ could effectively contribute to the proliferation of the stem cells cultured using serum free media.

We evaluated the possible mechanisms by which EMortal™ elicit its activating effects on the proliferation of human adult stem cells. We evaluated changes in the cytokines expression profiles in the cells in response to treatment with it using a human cytokine analysis kit. Specifically, we measured the levels of EGF, FGF-2, Flt-3 ligand, G-CSF, GM-CSF, TGF-β1, IL-3, IL-6, IL-8, TNF-α, and VEGF in the cells treated with it. We found that the production of VEGF, TGF-β1, and IL-6 was significantly increased by the treatment.

The level of TGF-β1 expression increased in this system expectedly. Other two cytokines, Vascular Endothelial Growth Factor(VEGF) and Interleukin-6(IL-6) also increased.

VEGF was originally identified in tumor biology. Tumors grow very large very rapidly and to do this they need a lot of nutrients. To ensure a good supply, tumor cells may produce VEGF to induce blood vessels in surrounding healthy tissue to grow into the tumor. VEGF production and increased angiogenesis can also see in wound healing and in some diseases such as psoriasis. However, VEGF is also produced by normal, healthy cells in various organs to maintain a good blood supply.

IL-6 is involved in the growth and differentiation of numerous cell types. In the skin it is produced primarily by keratinocytes. IL-6 is involved in barrier repair after skin injury. The IL-6 regulates epidermal permeability barrier homeostasis.

Collectively, these findings indicate that TGF-β1, VEGF and IL-6 are involved in the proliferation of cell induced by EMortal™.